This invention relates to an immunoassay device to be used for detecting an antigen or antibody in a specimen, and an immunoassay method using the same.
This invention also relates to an immunoassay method which comprises adding a specimen to an immunoassay device in which a labeling reagent zone comprising a labeled genetic recombinant syphilis treponeme (Treponema pallidum, hereinafter referred to as xe2x80x9cTPxe2x80x9d) antigen and a detection zone in which a TP antigen is immobilized to a matrix which can transfer a solution by capillarity are provided on the matrix; and detecting an anti-syphilis treponeme antibody (hereinafter referred to as xe2x80x9cthe anti-TP antibodyxe2x80x9d) in the specimen which is bound to the detection zone, and an immunoassay device to be used for said assay.
As a device which detects an antigen or antibody in a specimen simply and easily, an immunoassay device has been used. A conventional immunoassay device is schematically shown in FIG. 11 (see Japanese Provisional Patent Publication No. 47894/1978). The conventional immunoassay device has a membrane portion 20 comprising a material such as cellulose, and a developing solution-supplying portion 22 and an absorption portion 24 each comprising a water-absorbable material, provided at both ends of the membrane portion 20, respectively. In the membrane portion 20, a labeled substance dotting portion 26 to which a labeled substance labeled with a radioisotope is dotted is provided. Further, a detection line 28 is provided between the labeled substance dotting portion 26 and the absorption portion 24. At the detection line 28, an antibody or antigen which reacts with an antigen or antibody to be detected is immobilized to a membrane. Further, between the labeled substance dotting portion 26 and the developing solution-supplying portion 22, a specimen dotting portion 29 is provided.
When the device is used, a specimen solution is added to the specimen dotting portion 29, and a developing solution is added to the developing solution-supplying portion 22. The developing solution is developed from the developing solution-supplying portion 22 and reaches to the specimen dotting portion 29. The specimen dotted to the specimen dotting portion 29 is flowed by the developing solution flowing from the developing solution-supplying portion 22, reaches to the labeled substance dotting portion 26 and reacts with the labeled substance. When the reaction mixture is further flowed by the developing solution and reaches to the detection line 28, an antigen or antibody in the specimen is trapped by an antibody or antigen immobilized to the detection line 28 and held there. In this case, the antigen or antibody in the specimen is bound to the labeled substance so that the labeled substance is also held at the detection line 28. Therefore, when the antigen or antibody to be detected is contained in the specimen solution, the signal of the radioisotope is observed on the detection line 28. The developing solution and other components which are not trapped are absorbed into the absorption portion 24. On the other hand, when the antigen or antibody to be detected is not contained in the specimen solution, the labeled substance is not trapped at the detection line 28 and therefore is absorbed as such into the absorption portion 24, whereby the signal is not observed on the detection line 28. Thus, whether or not the antigen or antibody to be detected is contained in the specimen can be found by whether or not the signal of the radioisotope is detected at the detection line 28.
However, in the conventional immunoassay device as described above, there are problems that since the specimen dotted to the specimen dotting portion 29 is diluted with the developing solution during measurement, lowering of detection sensitivity is caused, and since the labeled substance is dissolved in the developing solution during development and reacts with the specimen, when an object to be measured has low concentration, in order to carry out an accurate test, a long reaction time is required to be taken.
An immunoassay device using a color latex has been also known. In this device, color latex particles to which an antibody or antigen which reacts with an antigen or antibody to be detected is bound are used as a labeled substance. This device is schematically shown in FIG. 4. A labeled substance dotting portion 32 is provided at one end of a membrane portion 30, and the above labeled substance is contained in the labeled substance dotting portion 32. On the other hand, to a detection line 36 is immobilized an antibody or antigen which reacts with an antigen or antibody to be detected. A specimen solution is added to a sample dotting portion 34 in the labeled substance dotting portion 32. The specimen solution and the labeled substance dissolved by the specimen solution are flowed while they are reacted, and when an antigen or antibody to be detected is contained in the specimen solution, the labeled substance is trapped at the detection line 36 as in the above case. The labeled substance contains a color latex so that the detection line 36 is colored. On the other hand, when the antigen or antibody to be detected is not contained in the specimen solution, the labeled substance is not trapped at the detection line 36 so that the detection line 36 is not colored. Thus, whether or not the antigen or antibody to be detected is contained in the specimen solution can be found by whether or not the detection line 36 is colored.
However, in the conventional immunoassay devices, there is a problem that a result which was negative at the time of judgment may be changed to be positive with a lapse of time. Therefore, when plural specimens are judged under the same conditions, it is necessary to carry out judgment at a certain judgment time or carry out judgment after a reaction is terminated by adding a reaction-terminating solution after a lapse of a certain period of time. However, when a large number of specimens are tested in parallel as in clinical tests, it is difficult to carry out judgment of all specimens at the same judgment time or add a reaction-terminating solution after a lapse of the same period of time. Further, when a reaction-terminating solution is added, the number of steps is increased to make an operation troublesome.
It is important to analyze living body components or drugs contained in blood, urine and the like for diagnosis of conditions of diseases and judgment of progress after therapy. As a method for analyzing these living body components, drugs and the like from a specimen simply and easily by utilizing an antigen-antibody reaction, there has been found a method of using a strip assay device comprising a strip-shaped filter paper impregnated with a reaction reagent. This assay method is a method in which a specimen is added to a specimen dotting zone provided on the filter paper of the device, a solution is developed and diagnosis is carried out from the degree of coloring shown at a detection zone provided on the filter paper. In the above strip assay device, a filter paper containing a necessary reagent (an enzyme-labeled antibody, a substrate, a coloring reagent or the like) depending on the system of a reaction is used, and analysis is carried out from coloring thereof so that a simple and easy method can be carried out without using a special judgment device. Also, there has been known an assay method of using a color latex or particles of metal colloid or the like as a labeled substance. In this assay method, analysis is carried out by using a reagent in which an antibody or antigen which reacts with an antigen or antibody to be detected is bound to particles and detecting the image of the particles bound to a detection zone.
In the prior art, as a method for detecting the anti-TP antibody, there have been known a method of using a TPHA reagent in which a TP cell component is bound to hemocytes, a method of using an indirect agglutination immunoassay reagent produced by binding a TP antigen to artificial particles and a method of using an immunoassay reagent comprising a TP antigen-binding solid phase and a labeled anti-globulin antibody. In all of these methods, 2 hours or longer is required from starting measurement to obtaining a result, and a measurement apparatus is used for judgment so that the methods cannot be used for an emergency test and measurement at a bedside within a short period of time.
In a method for measuring the anti-TP antibody using a conventional strip assay device, a reaction occurs between large amounts of globulin and a labeled antibody existing in a specimen, whereby measurement cannot be carried out. Therefore, there have been found a method of using a device in which a washing reservoir is added to an assay device (Japanese Provisional Patent Publication No. 126832/1993) and a method of using a device in which a zone for preventing signals generated by a labeled substance is provided in a developing solution (PCT Provisional Patent Publication No. 503439/1989).
In these methods, operations are complicated, and it is difficult to carry out measurement operations under constant conditions at all times. Further, due to influence of globulin or the like contained in a specimen, the methods are not satisfactory for carrying out measurement with good sensitivity. In order to solve such problems, when measurement of the anti-TP antibody in a specimen is carried out, it has been attempted to detect said antibody by using a TP antigen and a labeled TP antigen bound to a solid phase according to the sandwich method. However, it was not easy to introduce a labeled substance into a mixture containing a TP antigen obtained by culturing in a living body in the prior art, and a labeled antigen reagent which was substituted by a constant labeled substance at all times could not be obtained continuously. As described above, a method of using a labeled TP antigen has not been known.
In a conventional measurement method for the anti-TP antibody, there are problems that an operation is complicated and analysis with high precision cannot be carried out within a short period of time.
A first object of the present invention is to provide an immunoassay device in which detection sensitivity is high and an accurate test can be carried out within a shorter period of time as compared with a conventional immunoassay device, and an immunoassay method using the same.
A second object of the present invention is to provide an immunoassay device in which a judgment result which was negative at the time of judgment is not changed to be positive with a lapse of time, and an immunoassay method using the same.
A third object of the present invention is to provide an assay method by which a result can be obtained within a short period of time by a simple and easy operation as compared with a conventional method, and a device for practicing said assay.
The present inventors have studied intensively and consequently found that low sensitivity of a conventional assay device is caused by problems that a specimen dotted to a specimen dotting portion is diluted before it reacts with a labeled substance, a reaction of a labeled substance dotted to a labeled substance dotting portion and a specimen dotted to a specimen dotting portion is not carried out sufficiently, and the amount of a specimen solution dotted to a specimen dotting portion is small, and further found that these problems can be solved by incorporating a labeled substance on a membrane, to accomplish the present invention.
That is, the present invention provides an immunoassay device which comprises a labeled substance dotting portion and a specimen dotting portion provided thereon.
Also, the present inventors have studied intensively and consequently found that the above second object can be achieved not by incorporating a substrate of an enzyme into a developing solution as in the prior art, but by incorporating a substrate of an enzyme into a membrane in a dry state, to accomplish the present invention.
That is, the present invention provides an immunoassay device, comprising: a membrane portion; a labeled substance dotting portion provided on the membrane portion, wherein the labeled substance dotting portion comprises a pad of an absorbable material which contains a labeled substance and wherein the labeled substance is in mobilizable/diffusively bound form in the pad; a specimen dotting portion provided on the labeled substance dotting portion; a developing solution-supplying portion having a breakable solution reservoir, wherein the breakable solution reservoir contains a developing solution; a water-absorbable pad, wherein the developing solution-supplying portion and the water-absorbable pad are at opposite ends of the membrane portion; and a detection portion provided between the labeled substance dotting portion and the water absorbable pad, wherein an antibody or antigen is immobilized in the detection portion; wherein the labeled substance dotting portion is provided between the detection portion and the developing solution-supplying portion and yet at a position where a ratio LX/LT is less than 0.5, wherein LX is the distance from the longitudinal center of said labeled substance dotting portion to an end of said water-absorbable pad, the end of which is present at an upstream side of the solution transferring direction of the developing solution, and LT is the distance from said end of said water-absorbable pad to an end of said developing solution-supplying portion, the end of which is present at a downstream side of the solution transferring direction of the developing solution. Further, the present invention provides an immunoassay method using the above assay device of the present invention.
The present inventors have studied intensively and consequently found an immunoassay method in which a specimen is added to an immunoassay device for an anti-syphilis treponeme antibody, in which a labeling reagent zone comprising a labeled genetic recombinant TP antigen and a detection zone in which a TP antigen is immobilized to a matrix which can transfer a solution by capillarity are provided on the matrix; and an anti-syphilis treponeme antibody in the specimen bound to the detection zone is detected, and a novel immunoassay device for the anti-TP antibody, in which a labeling reagent zone comprising a labeled genetic recombinant TP antigen is provided on a matrix which can transfer a solution by capillarity, to accomplish the present invention. More specifically, they have found an immunoassay method which comprises using an immunoassay device for the anti-TP antibody, in which a developing solution zone containing a substrate, a labeling reagent zone comprising a genetic recombinant TP antigen labeled with an enzyme, a detection zone in which a TP antigen is immobilized to a matrix which can transfer a solution by capillarity, a specimen dotting zone and a developing solution-absorbing zone are provided on the matrix; dotting a specimen solution to the specimen dotting zone; supplying a developing solution to the developing solution zone; and detecting the enzyme immobilized to the detection zone in an amount depending on the anti-TP antibody in the specimen solution, with the substrate, and an immunoassay method which comprises using an immunoassay device for the anti-TP antibody, in which a specimen developing solution zone for adding a developing solution containing a specimen and a genetic recombinant TP antigen labeled with a radioisotope, a latex, metal colloid particles, fluorescent particles or colored particles, a detection zone in which a TP antigen is immobilized to a matrix which can transfer a solution by capillarity and a developing solution-absorbing zone are provided on the matrix; adding a specimen solution to the specimen dotting zone; supplying a developing solution to the developing solution zone; and detecting the labeled substance immobilized to the detection zone in an amount depending on the anti-TP antibody in the specimen solution.